The hyaluronan metabolism in the UV-irradiated human epidermis and the relevance of in vitro epidermal models.

Exposure to the sun affects the skin and may eventually result in UV-induced skin damage. It is generally known that hyaluronan (HA) is one of the main structural and functional components of the skin. However, UV-related changes in the HA metabolism in the skin have not yet been elucidated. Using qRT-PCR, confocal microscopy and LC-MS/MS we compared the naturally sun-exposed (SE), sun-protected, experimentally repeatedly UVA + UVB-exposed and acutely (once) UVA + UVB irradiated skin of Caucasian women. The epidermis was harvested by means of suction blistering 24 h after the acute irradiation. In addition, the epidermis was compared with a UV-irradiated in vitro reconstituted 3D epidermis (EpiDerm) and an in vitro 2D culture of normal human keratinocytes (NHEK). The amount of HA was found to be statistically significantly enhanced in the acutely irradiated epidermis. The acute UV evinced the upregulation of HA synthases (HAS2 and HAS3), hyaluronidases (HYAL2 and HYAL3), Cluster of differentiation 44 (CD44), and Cell Migration Inducing Proteins (CEMIP and CEMIP2), while only certain changes were recapitulated in the 3D epidermis. For the first time, we demonstrated the enhanced gene and protein expression of CEMIP and CEMIP2 following UV irradiation in the human epidermis. The data suggest that the HA metabolism is affected by UV in the irradiated epidermis and that the response can be modulated by the underlying dermis.

Octenidine with hyaluronan dressing versus a silver dressing in hard-to-heal wounds: a post-marketing study.

OBJECTIVE: This study was conducted to provide comparative data on the clinical efficacy and safety of a novel hard-to-heal wound dressing (Sorelex; Contipro a.s., Czech Republic) that combines octenidine and hyaluronan, compared with a silver-based dressing. METHOD: This multicentre, open-label, randomised, post-market, clinical follow-up study provides a comparison of a octenidine and hyaluronan combination with a standard-of-care (SoC) silver-containing dressing. The investigators evaluated the management of infection in the hard-to-heal wounds based on recorded changes in the appearance of the wound bed tissue and the presence of clinical signs of infection after three weeks of treatment. Other relevant assessed parameters of wound healing were: wound size; exudation level; wound pain level; and surrounding skin appearance. RESULTS: The study cohort included 48 patients in the Sorelex arm and 39 patients in the SoC arm. Both products evinced numerous parameters of wound infection management: reductions in the wound bed slough; marked decreases in wound size; the formation of re-epithelialisation and granulation tissue; and improved pain management. Sorelex significantly improved the condition of surrounding skin after three weeks of treatment, unlike SoC. Sorelex reduced wound area significantly more than SoC (p=0.04). No statistically significant differences were detected in other assessed parameters between the two study arms. All the participating investigators expressed their satisfaction with both products. No adverse reactions to Sorelex were recorded over the mean treatment period of 53 days. CONCLUSION: The octenidine and hyaluronan combination provides a new alternative choice of dressing for the treatment of infected hard-to-heal wounds when compared with a silver-based product.

Hyaluronan: A key player or just a bystander in skin photoaging?

Photoaged skin exhibits signs of inflammation, DNA damage and changes in morphology that are visible at the macroscopic and microscopic levels. Photoaging also affects the extracellular matrix (ECM) including hyaluronan (HA), the main polysaccharide component thereof. HA is a structurally simple but biologically complex molecule that serves as a water-retaining component and provides both a scaffold for a number of the proteins of the ECM and the ligand for cellular receptors. The study provides an overview of the literature concerning the changes in HA amount, size and metabolism, and the potential role of HA in photoaging. We also suggest novel HA contributions to photoaging based on our knowledge of the role of HA in other pathological processes, including the senescence and inflammation-triggered ECM reorganization. Moreover, we discuss potential direct or indirect intervention to mitigate photoaging that targets the hyaluronan metabolism, as well as supplementation.

Silver distribution in chronic wounds and the healing dynamics of chronic wounds treated with dressings containing silver and octenidine.

Although silver is an efficient antimicrobial and is a widely used antiseptic in wound healing, previous studies have reported the cytotoxic in vitro effects of silver dressings. Moreover, few studies have addressed the distribution of silver in chronic wounds. The study compares the healing of chronic wounds treated with a standard-of-care silver dressing (Ag-CMC) and a dressing containing antiseptic octenidine (OCT-HA). Biopsies were taken from two wound areas before the commencement of treatment (baseline), after 2 weeks and after 6 weeks (the end of the study). We analyzed the histopathologic wound-healing score, silver distribution, and expression of selected genes. The wound-healing score improved significantly in the wounded area treated with OCT-HA after 2 weeks compared to the baseline and the Ag-CMC. The Ag-CMC wound areas improved after 6 weeks compared to the baseline. Moreover, collagen maturation and decreases in the granulocyte and macrophage counts were faster in the OCT-HA parts. Treatment with OCT-HA resulted in less wound slough. The silver, visualized via autometallography, penetrated approximately 2 mm into the wound tissue and associated around capillaries and ECM fibers, and was detected in phagocytes. The metallothionein gene expression was elevated in the Ag-CMC wound parts. This exploratory study determined the penetration of silver into human chronic wounds and changes in the distribution thereof during treatment. We observed that silver directly affects the cells in the wound and elevates the metallothionein gene expression. Octenidine and hyaluronan dressings provide a suitable alternative to silver and carboxymethyl cellulose dressings without supplying silver to the wound.

The effect of hyperbaric oxygen therapy on gene expression: microarray analysis on wound healing.

Background: Hyperbaric oxygen (HBO2) therapy can have a positive effect on wound healing, angiogenesis and blood flow. No prior study has described the effects of HBO2 therapy and gene expression of this process. The goal of our research was to show the effects of HBO2 and its impact at the molecular level on angiogenesis, proliferation, differentiation, oxidative stress, inflammation, and extracellular matrix formation. Live animal subjects were used for simulating the process of wound healing under standard conditions and under the influence of HBO2. Methods: Two experimental groups were created using injured rabbits (N=24), one group (N=12) treated with hyperbaric therapy twice a day and one (N=12) with standard wound care management. Wounds were surgical, uninfected, and in healthy animal test subjects. We compared the whole genomic analysis of the transcriptome with the use of microarray technology at three intervals during treatment. Results: The induction of the wounds in rabbit skin increased expression of hundreds of genes in both treatment groups. The numbers of elevated and decreased genes gradually reduced as the wound healed. Gene expression analysis showed elevated expression of several genes associated with inflammation in both groups of injured animals. Genes connected to the process of angiogenesis, proliferation, differentiation, oxidative stress and extracellular matrix formation were without statistically significant changes. Conclusion: The evidence did not support that HBO2 had any significant effect on gene expression during wound healing. Additionally, there was no evidence to support that there were changes in gene expression in either treatment group.

A Potent Autophagy Inhibitor (Lys05) Enhances the Impact of Ionizing Radiation on Human Lung Cancer Cells H1299.

Autophagy inhibition through small-molecule inhibitors is one of the approaches to increase the efficiency of radiotherapy in oncological patients. A new inhibitor-Lys05-with the potential to accumulate within lysosomes and to block autophagy was discovered a few years ago. Several studies have addressed its chemosensitizing effects but nothing is known about its impact in the context of ionizing radiation (IR). To describe its role in radiosensitization, we employed radioresistant human non-small cell lung carcinoma cells (H1299, p53-negative). Combined treatment of H1299 cells by Lys05 together with IR decreased cell survival in the clonogenic assay and real-time monitoring of cell growth more than either Lys05 or IR alone. Immunodetection of LC3 and p62/SQSTM1 indicated that autophagy was inhibited, which correlated with increased SQSTM1 and decreased BNIP3 gene expression determined by qRT-PCR. Fluorescence microscopy and flow cytometry uncovered an accumulation of lysosomes. Similarly, transmission electron microscopy demonstrated the accumulation of autophagosomes confirming the ability of Lys05 to potentiate autophagy inhibition in H1299 cells. We report here for the first time that Lys05 could be utilized in combination with IR as a promising future strategy in the eradication of lung cancer cells.

A porcine model of skin wound infected with a polybacterial biofilm.

A clinically relevant porcine model of a biofilm-infected wound was established in 10 minipigs. The wounds of six experimental animals were infected with a modified polymicrobial Lubbock chronic wound biofilm consisting of Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa and Bacillus subtilis. Four animals served as uninfected controls. The wounds were monitored until they had healed for 24 days. The biofilm persisted in the wounds up to day 14 and significantly affected healing. The control to infected healed wound area ratios were: 45%/21%, 66%/37%, and 90%/57% on days 7, 10 and 14, respectively. The implanted biofilm prolonged inflammation, increased necrosis, delayed granulation and impaired development of the extracellular matrix as seen in histological and gene expression analyses. This model provides a therapeutic one-week window for testing of anti-biofilm treatments and for research on the pathogenesis of wound infections in pig that is clinically the most relevant animal wound healing model.

Low, medium, and high molecular weight hyaluronic acid effects on human dental pulp stem cells in vitro.

Hyaluronic acid (HA), an extracellular biopolymer found throughout the human body, holds promise as a biocompatible and biodegradable scaffold material. High molecular weight (HMW) HA degrades, generating low molecular weight (LMW) fragments with distinct properties. These fragments can influence the behaviour of cells, including human dental pulp stem cells (hDPSCs) incorporated into HA-containing hydrogels or scaffolds. Therefore, a comprehensive examination of the impact of a range of HA molecular weights on hDPSCs is essential before designing HA-based scaffolds for these cells. hDPSC lines were cultured with LMW HA (800 Da, 1600 Da, 15 kDa), medium molecular weight HA (237 kDa), or HMW HA (1500 kDa) over six passages. The various molecular weights had negligible effects on hDPSCs viability, morphology, adhesion, or relative telomere length. Furthermore, the expression of key surface stemness markers (CD29, CD44, CD73, CD90) remained unaltered. HA did not induce osteogenic, chondrogenic, or adipogenic differentiation. Moreover, the potential for chondrogenic and osteogenic differentiation was not adversely affected by LMW or HMW HA. Various molecular weights of HA seem safe, biocompatible and therefore suitable components for hDPSCs-containing scaffolds. These findings affirm that the hDPCSs will not be negatively affected by HA fragments resulting from scaffold degradation.

Phosphorylation of tyrosine 90 in SH3 domain is a new regulatory switch controlling Src kinase.

The activation of Src kinase in cells is strictly controlled by intramolecular inhibitory interactions mediated by SH3 and SH2 domains. They impose structural constraints on the kinase domain holding it in a catalytically non-permissive state. The transition between inactive and active conformation is known to be largely regulated by the phosphorylation state of key tyrosines 416 and 527. Here, we identified that phosphorylation of tyrosine 90 reduces binding affinity of the SH3 domain to its interacting partners, opens the Src structure, and renders Src catalytically active. This is accompanied by an increased affinity to the plasma membrane, decreased membrane motility, and slower diffusion from focal adhesions. Phosphorylation of tyrosine 90 controlling SH3-medited intramolecular inhibitory interaction, analogical to tyrosine 527 regulating SH2-C-terminus bond, enables SH3 and SH2 domains to serve as cooperative but independent regulatory elements. This mechanism allows Src to adopt several distinct conformations of varying catalytic activities and interacting properties, enabling it to operate not as a simple switch but as a tunable regulator functioning as a signalling hub in a variety of cellular processes.

An in vitro model that mimics the foreign body response in the peritoneum: Study of the bioadhesive properties of HA-based materials.

A cascade of reactions known as the foreign body response (FBR) follows the implantation of biomaterials leading to the formation of a fibrotic capsule around the implant and subsequent health complications. The severity of the FBR is driven mostly by the physicochemical characteristics of implanted material, the method and place of implantation, and the degree of immune system activation. Here we present an in vitro model for assessing new materials with respect to their potential to induce a FBR in the peritoneum. The model is based on evaluating protein sorption and cell adhesion on the implanted material. We tested our model on the free-standing films prepared from hyaluronan derivatives with different hydrophobicity, swelling ratio, and rate of solubilization. The proteomic analysis of films incubated in the mouse peritoneum showed that the presence of fibrinogen was driving the cell adhesion. Neither the film surface hydrophobicity/hydrophilicity nor the quantity of adsorbed proteins were decisive for the induction of the long-term cell adhesion leading to the FBR, while the dissolution rate of the material proved to be a crucial factor. Our model thus helps determine the probability of a FBR to materials implanted in the peritoneum while limiting the need for in vivo animal testing.

The Degradation of Hyaluronan in the Skin.

Hyaluronan (HA) comprises a fundamental component of the extracellular matrix and participates in a variety of biological processes. Half of the total amount of HA in the human body is present in the skin. HA exhibits a dynamic turnover; its half-life in the skin is less than one day. Nevertheless, the specific participants in the catabolism of HA in the skin have not yet been described in detail, despite the essential role of HA in cutaneous biology. A deeper knowledge of the processes involved will act to support the development of HA-based topical and implantable materials and enhance the understanding of the various related pathological cutaneous conditions. This study aimed to characterize the distribution and activity of hyaluronidases and the other proteins involved in the degradation of HA in healthy human full-thickness skin, the epidermis and the dermis. Hyaluronidase activity was detected for the first time in healthy human skin. The degradation of HA occurred in lysates at an acidic pH. HA gel zymography revealed a single band corresponding to approximately 50 kDa. This study provided the first comprehensive view of the distribution of canonic HA-degrading proteins (HYAL1 and HYAL2) in human skin employing IHF and IHC. Furthermore, contrary to previous assumptions TMEM2, a novel hyaluronidase, as well as CEMIP, a protein involved in HA degradation, were localized in the human epidermis, as well as in the dermis.

Retinoic Acid Grafted to Hyaluronic Acid Activates Retinoid Gene Expression and Removes Cholesterol from Cellular Membranes.

All-trans-retinoic acid (atRA) is a potent ligand that regulates gene expression and is used to treat several skin disorders. Hyaluronic acid (HA) was previously conjugated with atRA (HA-atRA) to obtain a novel amphiphilic compound. HA-atRA forms micelles that incorporate hydrophobic molecules and facilitate their transport through the skin. The aim of this study was to determine the influence of HA-atRA on gene expression in skin cells and to compare it with that of unbound atRA. Gene expression was investigated using microarrays and a luciferase system with a canonical atRA promoter. HA-atRA upregulated gene expression similarly to atRA. However, HA-atRA activated the expression of cholesterol metabolism genes, unlike atRA. Further investigation using HPLC and filipin III staining suggested that the treated cells induced cholesterol synthesis to replenish the cholesterol removed from the cells by HA-atRA. HA modified with oleate (HA-C18:1) removed cholesterol from the cells similarly to HA-atRA, suggesting that the cholesterol removal stemmed from the amphiphilic nature of the two derivatives. HA-atRA induces retinoid signaling. Thus, HA-atRA could be used to treat skin diseases, such as acne and psoriasis, where the combined action of atRA signaling and anti-inflammatory cholesterol removal may be potentially beneficial.

Retinoic acid grafted to hyaluronan for skin delivery: Synthesis, stability studies, and biological evaluation.

All-trans retinoic acid (ATRA) was grafted to hyaluronan (HA) via esterification. The reaction was mediated by mixed anhydrides. A perfect control of the degree of substitution (0.5-7.5%) was obtained by varying the molar ratio of retinoic acid in the feed. The degree of substitution plays a significant role in the long-term stability. The photodegradation of HA-ATRA upon UVA irradiation resulted in ß-ionone, ß-cyclocitral and 5,6-epoxy-(E)-retinoic acid. The photostability of the conjugate had increased with the combination with morin. The chemical structure of HA-ATRA and its degradation products was elucidated using NMR spectroscopy, SEC-MALLS, and gas chromatography-mass spectrometry (GC-MS). ATRA did not loss its biological activity after conjugation, as demonstrated by gene expression. The derivative was able to penetrate across the stratum corneum. Besides, HA-ATRA downregulated the expression of anti-inflammatory interleukins 6 and 8. HA-ATRA would be expected to be used for transdermal drug delivery or cosmetics.

Effects of wound dressings containing silver on skin and immune cells.

Wound dressings with silver have been shown to be cytotoxic in vitro. However, the extrapolation of this cytotoxicity to clinical settings is unclear. We applied dressings with various forms of silver on porcine skin ex vivo and investigated silver penetration and DNA damage. We assessed antimicrobial efficacy, cytotoxicity to skin cells, and immune response induced by the dressings. All dressings elevated the DNA damage marker γ-H2AX and the expression of stress-related genes in explanted skin relative to control. This corresponded with the amount of silver in the skin. The dressings reduced viability, induced oxidative stress and DNA damage in skin cells, and induced the production of pro-inflammatory IL-6 by monocytes. The oxidative burst and viability of activated neutrophils decreased. The amount of silver released into the culture medium varied among the dressings and correlated with in vitro toxicity. However, antimicrobial efficiencies did not correlate strongly with the amount of silver released from the dressings. Antimicrobial efficiency and toxicity are driven by the form of silver and the construction of dressings and not only by the silver concentration. The damaging effects of silver dressings in ex vivo skin highlight the importance of thorough in vivo investigation of silver dressing toxicity.

Dual role of iodine, silver, chlorhexidine and octenidine as antimicrobial and antiprotease agents.

OBJECTIVES: The majority of human chronic wounds contain bacterial biofilms, which produce proteases and retard the resolution of inflammation. This in turn leads to elevated patient protease activity. Chronic wounds progressing towards closure show a reduction in proteolytic degradation. Therefore, the modulation of protease activity may lead to the faster healing of chronic wounds. Antimicrobials are used to control biofilm-based infection; however, some of them also exhibit the inhibition of matrix metalloproteinases and bacterial proteases. We investigated the antimicrobial agents used in wound healing for their potential to inhibit bacterial and host proteases relevant to chronic wounds. METHODS: Using in vitro zymography, we tested the ability of povidone-iodine, silver lactate, chlorhexidine digluconate, and octenidine hydrochloride to inhibit selected human proteases and proteases from Pseudomonas aeruginosa, Staphylococcus aureus, Serratia marcescens, and Serratia liquefaciens. We investigated penetration and skin protease inhibition by means of in situ zymography. RESULTS: All the tested antimicrobials inhibited both eukaryotic and prokaryotic proteases in a dose-dependent manner in vitro. The tested compounds were also able to penetrate into skin ex vivo and inhibit the resident proteases. Silver lactate and chlorhexidine digluconate showed an inhibitory effect ex vivo even in partial contact with skin in Franz diffusion cells. CONCLUSIONS: Our in vitro and ex vivo results suggest that wound healing devices which contain iodine, silver, chlorhexidine, and octenidine may add value to the antibacterial effect and also aid in chronic wound healing. Antiprotease effects should be considered in the design of future antimicrobial wound healing devices.

Novel FRET-Based Src Biosensor Reveals Mechanisms of Src Activation and Its Dynamics in Focal Adhesions.

Src kinase plays an important role in a multitude of fundamental cellular processes and is often found deregulated in tumors. Active Src adopts an open conformation, whereas inactive Src is characterized by a very compact structure stabilized by inhibitory intramolecular interactions. Taking advantage of this spatial regulation, we constructed a fluorescence resonance energy transfer (FRET)-based Src biosensor and analyzed conformational changes of Src following Src activation and the spatiotemporal dynamics of Src activity in cells. We found that activatory mutations either in regulatory or kinase domains induce opening of the Src structure. Surprisingly, we discovered that Src inhibitors differ in their effect on the Src structure, some counterintuitively inducing an open conformation. Finally, we analyzed the dynamics of Src activity in focal adhesions by FRET imaging and found that Src is rapidly activated during focal adhesion assembly, and its activity remains steady and high throughout the life cycle of focal adhesion and decreases during focal adhesion disassembly.

Linolenic acid grafted hyaluronan: Process development, structural characterization, biological assessing, and stability studies.

In this study, hyaluronan (HA) was grafted with alpha-linolenic acid (αLNA) by benzoyl mixed anhydrides methodology, which allowed the derivatization of HA under mild reaction conditions. The reaction was optimized and transferred from laboratory to semi-scale production. The derivative revealed an unexpected cytotoxicity after oven drying and storage at 40°C. For this reason, the storage conditions of sodium linolenyl hyaluronate (αLNA-HA) were optimized in order to preserve the beneficial effect of the derivative. Oven, spray dried and lyophilized samples were prepared and stored at -20°C, 4°C and 25°C up to 6 months. A comprehensive material characterization including stability study of the derivative, as well as evaluation of possible changes on chemical structure and presence of peroxidation products were studied by Nuclear magnetic resonance (NMR), Fourier transform infrared spectroscopy (FTIR), gas chromatography-mass spectrometry (GC-MS), thermogravimetric analysis (TGA) and complemented with assessment of in vitro viability on mouse fibroblasts NIH-3T3. The most stable αLNA-HA derivative was obtained after spray drying and storage at ambient temperature under inert atmosphere. The choice of inert atmosphere is recommended to suppress oxidation of αLNA supporting the positive influence of the derivative on cell viability. The encapsulation of hydrophobic drugs of αLNA-HA were also demonstrated.